Assay reagents for a neurogranin diagnostic kit

ABSTRACT

The present invention relates to the field of biomarkers. More specifically, the present invention relates to assay reagents useful in detecting neurogranin. In a specific embodiment, the present invention provides an isolated antibody or fragment thereof that specifically binds to neurogranin. In another embodiment, the present invention provides a polynucleotide aptamer that specifically binds neurogranin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/485,375, filed May 12, 2011, which is incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with U.S. government support under grant no.NHLBI 1 R01 HL091759-02, and NHLBI 5U54HL090515-02. The U.S. governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of biomarkers. Morespecifically, the present invention relates to assay reagents useful indetecting biomarkers.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“P11546-02_Sequence_Listing_ST25.txt.” The sequence listing is 3,146bytes in size, and was created on May 10, 2012. It is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Brain injuries are complex and can have multiple severe clinicaloutcomes. Injury of the brain and spinal cord can result from headtrauma, stroke, traumatic birth, heart surgery, cardiac arrest andpatients requiring cardiovascular support with ventricular assistdevices or extracorporeal membrane oxygenation (ECMO). Moreover,detection of subclinical brain injury is difficult, especially inchildren and neonates with birth-related injury. In addition, childrenwith sickle cell disease are at high risk for subclinical brain injury.Untreated subclinical brain injuries in children can progress to overtstroke, neurological damage, learning problems and memory loss.

Unfortunately, clinical tools such as physical exam, and imaging (CTScan or MRI) are subjective, not widely available, not sensitive orspecific enough and or too costly to identify the infant, child or adultwith brain injury. There is a great clinical need to identify patientswith brain injury and especially subclinical injury because theseinfants, children and adults are at significant risk of progressing toovert stroke and development of cognitive and motor loss, and dementia.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the development ofaptamers and antibodies that bind neurogranin. In one embodiment, thepresent invention provides an isolated antibody or fragment thereof thatspecifically binds to neurogranin. In a specific embodiment, theisolated antibody or fragment thereof specifically binds to amino acids1-78 of SEQ ID NO:4. In a more specific embodiment, an isolated antibodyor fragment thereof specifically binds to amino acids 55-78 of SEQ IDNO:4. In certain embodiments, the antibody or fragment thereof ispolyclonal. In other embodiments, the antibody or fragment thereof ismonoclonal. In a specific embodiment, the antibody or fragment thereofis mammalian. The antibody or fragment thereof can be human. Inparticular embodiments, the present invention provides a hybridoma cellwhich produces the antibody or fragment described herein.

The antibody fragment can be selected from the group consisting of a Fabfragment; a F(ab′) 2 fragment; a Fv fragment; and a single chainfragment. The isolated antibody or fragment thereof can further comprisea detectable substance coupled to the antibody. In certain embodiments,the detectable substance is selected from the group consisting of anenzyme; a fluorescent label; a radioisotope; and chemiluminescent label.

In one embodiment, the isolated antibody or fragment thereofspecifically binds to neurogranin in an ELISA. In another embodiment,the isolated antibody or fragment thereof specifically binds toneurogranin in a competitive-binding assay. In yet another embodiment,the isolated antibody or fragment thereof specifically binds toneurogranin in a radioimmunoassay. In a further embodiment, the isolatedantibody or fragment thereof specifically binds to neurogranin in afluorescence-activated cell sorting (FACS) assay.

In another aspect, the present invention provides kits useful fordetecting neurogranin. A kit for detecting neurogranin may comprise (a)an isolated antibody described herein; and (b) at least one component todetect binding of the isolated antibody to neurogranin.

In another embodiment, the present invention provides an isolatedantibody obtained from an animal that has been immunized withneurogranin, wherein the antibody specifically binds to an antigenicepitope-bearing polypeptide fragment of neurogranin. In an specificembodiment, the present invention provides a monoclonal antibodydesignated 30.5.2 that specifically binds neurogranin. In anotherembodiment, the present invention provides an anti-neurograninmonoclonal antibody which is produced by a hybridoma.

In another aspect, the present invention provides aptamers that bindneurogranin. In certain embodiments, the present invention provides apolynucleotide aptamer that specifically binds neurogranin. In anotherembodiment, the neurogranin is human neurogranin. In a specificembodiment, the aptamer binds to neurogranin with a Kd of less thanabout 1000 nM. In a more specific embodiment, the aptamer binds toneurogranin with a Kd of less than about 100 nM. In a furtherembodiment, the aptamer binds to neurogranin with a Kd of less thanabout 20 nM. The polynucleotide aptamer of the present invention canconsist of about 10 to about 100 nucleotides. In another embodiment, thepolynucleotide aptamer consists of about 20 to about 80 nucleotides. Inyet another embodiment, the polynucleotide aptamer consists of about 30to about 50 nucleotides. In particular embodiments, the polynucleotideaptamer is an RNA aptamer.

In a specific embodiment, the polynucleotide aptamer comprises anucleotide sequence at least 80% identical to any one of SEQ ID NOS: 4-6or a fragment thereof of at least ten contiguous nucleotides. In anotherembodiment, the polynucleotide aptamer comprises a nucleotide sequenceat least 90% identical to any one of SEQ ID NOS: 4-6 or a fragmentthereof of at least ten contiguous nucleotides. In yet anotherembodiment, the polynucleotide aptamer comprises a nucleotide sequenceat least 95% identical to any one of SEQ ID NOS: 4-6 or a fragmentthereof of at least ten contiguous nucleotides.

In another embodiment, a polynucleotide aptamer comprises the nucleotidesequence of any one of SEQ ID NOS: 4-6 or a fragment thereof of at leastten contiguous nucleotides. In a further embodiment, a polynucleotideaptamer consists of the nucleotide sequence of any one of SEQ ID NOS:4-6 or a fragment thereof of at least ten contiguous nucleotides. Thepolynucleotide aptamer can comprise at least one modifiedinternucleotide linker. In other embodiments, the polynucleotide aptamercan comprise at least one terminal blocker. The polynucleotide aptamerof the present invention can be linked to a conjugate.

The present invention further provides a polynucleotide encoding apolynucleotide aptamer described herein. In a specific embodiment, thepresent invention provides a vector comprising a polynucleotidedescribed herein. The present invention further provides a cellcomprising a polynucleotide aptamer described herein. A cell maycomprise two or more different polynucleotide aptamers. The presentinvention also provides a polynucleotide described herein, as well as avector described herein.

In another aspect, the present invention provides methods of diagnosinga disease or disorder associated with neurogranin in a subject. Incertain embodiments, the method comprises measuring the level ofneurogranin in the subject by binding neurogranin with a polynucleotideaptamer and determining the amount of aptamer bound to neurogranin. In aparticular embodiment, the binding occurs in a sample obtained from thesubject. In other embodiments, a method for determining the amount ofneurogranin in a sample comprises the step of detecting a peptidespecific to neurogranin using a triple quadrupole mass spectrometer andmultiple reaction monitoring, wherein the peptide specific toneurogranin comprises SEQ ID NO:7.

In a specific embodiment, the present invention provides a neurograninRNA aptamer (NRGN-A1) with a sequence comprising the sequence of:TCTAACGCCTCCCGTATGTTTTCCTTTTTCCATTGCGGAT (SEQ ID NO:4), which can bindto neurogranin protein and is useful for a detection assay. In anotherembodiment, the present invention provides a neurogranin RNA aptamer(NRGN-A6) with a sequence comprising the sequence of:TTTTCATTTTCATTTTTTTCCAAATCGATCCGCCGGACCTTAT (SEQ ID NO:6), which canbind to neurogranin protein and is useful for a detection assay.

In another embodiment, the present invention provides a monoclonalantibody identified as 30.5.2 to human neurogranin and is useful for adiagnostic detection assay for brain injury. In a further embodiment, amethod of using a neurogranin RNA aptamer in a diagnostic assay forbrain injury in a mammalian subject, comprises the steps of (a)obtaining a sample from the subject suspected of having a brain injury,and (b) performing an assay using the aptamer to detect a level ofneurogranin in the sample, wherein a level of neurogranin in the samplethat is significantly different than in a sample obtained from a controlsubject that does not have a brain injury is diagnostic of a braininjury. In yet another embodiment, a method of using a neurograninmonoclonal antibody in a diagnostic assay for brain injury in amammalian subject, comprises the steps of (a) obtaining a sample fromthe subject suspected of having a brain injury, and (b) performing anassay using the antibody to detect the level of neurogranin in thesample, wherein a level of neurogranin in the sample that issignificantly different than in a sample obtained from a control subjectthat does not have a brain injury is diagnostic of a brain injury. Insuch methods, the brain injury is selected from the group consisting ofsubclinical brain injury and overt brain injury. Finally, the presentinvention provides a diagnostic/prognostic kit for brain injurycomprising capture and detection reagents for neurogranin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a gel showing the minimum amount of neurogranin aptamersneeded to for detection of neurogranin protein.

FIG. 2 shows the results of a pull-down assay using the neurograninaptamers.

FIG. 3 displays the signals of neurogranin signature peptide and labeledstandard peptide using an ABI Sciex Qtrap 4000 triple quadrapole massspectrometer.

FIG. 4 shows His-NRGN on PAGE gel after coomassie staining. Thepredicted molecular weight of His-NRGN is 8.5 Kd.

FIG. 5 shows the standard curve of the direct ELISA for recombinant NRGNusing mouse monoclonal antibody 30.5.2. The concentration range is0.002-10 ng/ml.

FIG. 6 shows the standard curve of the direct ELISA for recombinant NRGNusing an anti-human monoclonal antibody to neurogranin. Theconcentration range is 75 ng/ml.

FIG. 7 shows neurogranin and GFAP levels from patients undergoingcardiopulmonary bypass for surgical repair of congenital heart disease.

FIG. 8 is a graph showing that neurogranin is a circulating biomarker ofacute brain injury and appears earlier in the circulation than glialfibrillary acidic protein, a known circulating biomarker of acutestroke.

FIG. 9A. is a log plot of plasma Neurogranin concentrations in childrenwith sickle cell disease (SCD) (n=115) and age-matched, non-SCD controls(n=46). The dashed line marks the 95th percentile value among 46 non-SCDcontrols. Bars represent median values. FIG. 9B is a log plot of plasmaNeurogranin concentrations in children with SCD and SCI (n=64), with SCDand no SCI (n=51), and age-matched, non-SCD controls (n=46). The dashedline marks the 95th percentile value among 60 healthy pediatriccontrols, and percentages list the proportions above the 95th percentileof healthy controls.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methods and components, etc., described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing particular embodiments only, and isnot intended to limit the scope of the present invention. It must benoted that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include the plural reference unless the contextclearly dictates otherwise. Thus, for example, a reference to a“protein” is a reference to one or more proteins, and includesequivalents thereof known to those skilled in the art and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Specific methods, devices, andmaterials are described, although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention.

All publications cited herein are hereby incorporated by referenceincluding all journal articles, books, manuals, published patentapplications, and issued patents. In addition, the meaning of certainterms and phrases employed in the specification, examples, and appendedclaims are provided. The definitions are not meant to be limiting innature and serve to provide a clearer understanding of certain aspectsof the present invention.

I. Definitions

The term “brain injury” refers to a condition in which the brain isdamaged by injury caused by an event. As used herein, an “injury” is analteration in cellular or molecular integrity, activity, level,robustness, state, or other alteration that is traceable to an event.For example, an injury includes a physical, mechanical, chemical,biological, functional, infectious, or other modulator of cellular ormolecular characteristics. An event can include a physical trauma suchas an impact (percussive) or a biological abnormality such as a strokeresulting from either blockade or leakage of a blood vessel. An event isoptionally an infection by an infectious agent. A person of skill in theart recognizes numerous equivalent events that are encompassed by theterms injury or event.

More specifically, the term “brain injury” refers to a condition thatresults in central nervous system damage, irrespective of itspathophysiological basis. Among the most frequent origins of a “braininjury” are stroke and traumatic brain injury. A “stroke” is classifiedinto hemorrhagic and non-hemorrhagic. Examples of hemorrhagic strokeinclude cerebral hemorrhage, subarachnoid hemorrhage, and intracranialhemorrhage secondary to cerebral arterial malformation, while examplesof non-hemorrhagic stroke include cerebral infarction.

The term “traumatic brain injury” or “TBI” refer to traumatic injuriesto the brain which occur when physical trauma causes brain damage. Forexample, TBI can result from a closed head injury or a penetrating headinjury. A “non-traumatic brain injury” refers to brain injuries that donot involve ischemia or external mechanical force (e.g., stroke,Alzheimer's disease, Parkinson's disease, Huntington's disease, multiplesclerosis, amyotrophic lateral sclerosis, brain hemorrhage, braininfections, brain tumor, and the like).

The term “brain injury” also refers to subclinical brain injury, spinalcord injury, and anoxic-ischemic brain injury. The term “subclinicalbrain injury” (SCI) refers to brain injury without overt clinicalevidence of brain injury. A lack of clinical evidence of brain injurywhen brain injury actually exists could result from degree of injury,type of injury, level of consciousness, and medications, particularlysedation and anesthesia.

The term “spinal cord injury” refers to a condition in which the spinalcord receives compression/detrition due to a vertebral fracture ordislocation to cause dysfunction. As used herein, the term“anoxic-ischemic brain injury” refers to deprivation of oxygen supply tobrain tissue resulting in compromised brain function and includescerebral hypoxia. For example, anoxic-ischemic brain injury includesfocal cerebral ischemia, global cerebral ischemia, hypoxic hypoxia(i.e., limited oxygen in the environment causes reduced brain function,such as with divers, aviators, mountain climbers, and fire fighters, allof whom are at risk for this kind of cerebral hypoxia), obstructions inthe lungs (e.g., hypoxia resulting from choking, strangulation, thecrushing of the windpipe).

As used herein, the term “comparing” refers to making an assessment ofhow the proportion, level or cellular localization of one or morebiomarkers in a sample from a patient relates to the proportion, levelor cellular localization of the corresponding one or more biomarkers ina standard or control sample. For example, “comparing” may refer toassessing whether the proportion, level, or cellular localization of oneor more biomarkers in a sample from a patient is the same as, more orless than, or different from the proportion, level, or cellularlocalization of the corresponding one or more biomarkers in standard orcontrol sample. More specifically, the term may refer to assessingwhether the proportion, level, or cellular localization of one or morebiomarkers in a sample from a patient is the same as, more or less than,different from or otherwise corresponds (or not) to the proportion,level, or cellular localization of predefined biomarker levels thatcorrespond to, for example, a patient having subclinical brain injury(SCI), not having SCI, is responding to treatment for SCI, is notresponding to treatment for SCI, is/is not likely to respond to aparticular SCI treatment, or having/not having another disease orcondition. In a specific embodiment, the term “comparing” refers toassessing whether the level of one or more biomarkers of the presentinvention in a sample from a patient is the same as, more or less than,different from other otherwise correspond (or not) to levels of the samebiomarkers in a control sample (e.g., predefined levels that correlateto uninfected individuals, standard SCI levels, etc.).

As used herein, the terms “indicates” or “correlates” (or “indicating”or “correlating,” or “indication” or “correlation,” depending on thecontext) in reference to a parameter, e.g., a modulated proportion,level, or cellular localization in a sample from a patient, may meanthat the patient has SCI. In specific embodiments, the parameter maycomprise the level of one or more biomarkers of the present invention. Aparticular set or pattern of the amounts of one or more biomarkers mayindicate that a patient has SCI (i.e., correlates to a patient havingSCI). In other embodiments, a particular set or pattern of the amountsof one or more biomarkers may be correlated to a patient beingunaffected (i.e., indicates a patient does not have SCI). In certainembodiments, “indicating,” or “correlating,” as used according to thepresent invention, may be by any linear or non-linear method ofquantifying the relationship between levels of biomarkers to a standard,control or comparative value for the assessment of the diagnosis,prediction of SCI or SCI progression, assessment of efficacy of clinicaltreatment, identification of a patient that may respond to a particulartreatment regime or pharmaceutical agent, monitoring of the progress oftreatment, and in the context of a screening assay, for theidentification of an anti-SCI therapeutic.

The terms “patient,” “individual,” or “subject” are used interchangeablyherein, and refer to a mammal, particularly, a human. The patient mayhave mild, intermediate or severe disease. The patient may be treatmentnaïve, responding to any form of treatment, or refractory. The patientmay be an individual in need of treatment or in need of diagnosis basedon particular symptoms or family history. In some cases, the terms mayrefer to treatment in experimental animals, in veterinary application,and in the development of animal models for disease, including, but notlimited to, rodents including mice, rats, and hamsters; and primates.

The terms “measuring” and “determining” are used interchangeablythroughout, and refer to methods which include obtaining a patientsample and/or detecting the level of a biomarker(s) in a sample. In oneembodiment, the terms refer to obtaining a patient sample and detectingthe level of one or more biomarkers in the sample. In anotherembodiment, the terms “measuring” and “determining” mean detecting thelevel of one or more biomarkers in a patient sample. Measuring can beaccomplished by methods known in the art and those further describedherein. The term “measuring” is also used interchangeably throughoutwith the term “detecting.”

The terms “sample,” “patient sample,” “biological sample,” and the like,encompass a variety of sample types obtained from a patient, individual,or subject and can be used in a diagnostic or monitoring assay. Thepatient sample may be obtained from a healthy subject, a diseasedpatient or a patient having associated symptoms of SCI. Moreover, asample obtained from a patient can be divided and only a portion may beused for diagnosis. Further, the sample, or a portion thereof, can bestored under conditions to maintain sample for later analysis. Thedefinition specifically encompasses blood and other liquid samples ofbiological origin (including, but not limited to, plasma, serum,peripheral blood, cerebrospinal fluid, urine, saliva, stool and synovialfluid), solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. In aspecific embodiment, a sample comprises a blood sample. In anotherembodiment, a serum sample is used. The definition also includes samplesthat have been manipulated in any way after their procurement, such asby centrifugation, filtration, precipitation, dialysis, chromatography,treatment with reagents, washed, or enriched for certain cellpopulations. The terms further encompass a clinical sample, and alsoinclude cells in culture, cell supernatants, tissue samples, organs, andthe like. Samples may also comprise fresh-frozen and/or formalin-fixed,paraffin-embedded tissue blocks, such as blocks prepared from clinicalor pathological biopsies, prepared for pathological analysis or study byimmunohistochemistry.

Various methodologies of the instant invention include a step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control,” referred to interchangeably herein as an“appropriate control” or a “control sample.” A “suitable control,”“appropriate control” or a “control sample” is any control or standardfamiliar to one of ordinary skill in the art useful for comparisonpurposes. In one embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.,determined in a cell, organ, or patient, e.g., a control or normal cell,organ, or patient, exhibiting, for example, normal traits. For example,the biomarkers of the present invention may be assayed for levels in asample from an unaffected individual (UI) or a normal control individual(NC) (both terms are used interchangeably herein). In anotherembodiment, a “suitable control” or “appropriate control” is a value,level, feature, characteristic, property, etc. determined prior toperforming a therapy (e.g., an SCI treatment) on a patient. In yetanother embodiment, a transcription rate, mRNA level, translation rate,protein level, biological activity, cellular characteristic or property,genotype, phenotype, etc., can be determined prior to, during, or afteradministering a therapy into a cell, organ, or patient. In a furtherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc. A“suitable control” can be a profile or pattern of levels of one or morebiomarkers of the present invention that correlates to SCI, to which apatient sample can be compared. The patient sample can also be comparedto a negative control, i.e., a profile that correlates to not havingSCI.

The term “isolated” designates a biological material (nucleic acid orprotein) that has been removed from its original environment (theenvironment in which it is naturally present). For example, apolynucleotide present in its natural state in a plant or an animal isnot isolated, however the same polynucleotide separated from theadjacent nucleic acids in which it is naturally present, is considered“isolated”. The term “purified” does not require the material to bepresent in a form exhibiting absolute purity, exclusive of the presenceof other compounds. It is rather a relative definition.

A “nucleic acid” or “polynucleotide” refers to the phosphate esterpolymeric form of ribonucleosides (adenosine, guanosine, uridine orcytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), orany phosphoester analogs thereof, such as phosphorothioates andthioesters, in either single stranded form, or a double-stranded helix.Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. Theterm nucleic acid molecule, and in particular DNA or RNA molecule,refers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear or circularDNA molecules (e.g., restriction fragments), plasmids, and chromosomes.In discussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

The term “fragment” will be understood to mean a nucleotide sequence ofreduced length relative to the reference nucleic acid and comprising,over the common portion, a nucleotide sequence identical to thereference nucleic acid. Such a nucleic acid fragment according to theinvention may be, where appropriate, included in a larger polynucleotideof which it is a constituent. Such fragments comprise, or alternativelyconsist of, oligonucleotides ranging in length from at least 6, 8, 9,10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51,54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200,300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a nucleicacid according to the invention.

The term “percent identity” or “percent identical,” as known in the art,is a relationship between two or more polypeptide sequences or two ormore polynucleotide sequences, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesmay be performed using the Clustal method of alignment (Higgins andSharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method may be selected: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include but is not limited to the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410(1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715USA). Within the context of this application it will be understood thatwhere sequence analysis software is used for analysis, that the resultsof the analysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters which originally load with thesoftware when first initialized.

The terms “specifically binds to,” “specific for,” and relatedgrammatical variants refer to that binding which occurs between suchpaired species as antibody/antigen, aptamer/target, enzyme/substrate,receptor/agonist and lectin/carbohydrate which may be mediated bycovalent or non-covalent interactions or a combination of covalent andnon-covalent interactions. When the interaction of the two speciesproduces a non-covalently bound complex, the binding which occurs istypically electrostatic, hydrogen-bonding, or the result of lipophilicinteractions. Accordingly, in certain embodiments, “specific binding”occurs between a paired species where there is interaction between thetwo which produces a bound complex having the characteristics of, forexample, an antibody/antigen or enzyme/substrate interaction. Inparticular, the specific binding is characterized by the binding of onemember of a pair to a particular species and to no other species withinthe family of compounds to which the corresponding member of the bindingmember belongs. Thus, for example, an antibody typically binds to asingle epitope and to no other epitope within the family of proteins. Insome embodiments, specific binding between an antigen and an antibodywill have a binding affinity of at least 10⁻⁶ M. In other embodiments,the antigen and antibody will bind with affinities of at least 10⁻⁷ M,10⁻⁸ M to 10⁻⁹ M, 10⁻¹⁰M, 10⁻¹¹ M, or 10⁻¹² M. In certain embodiments,the term refers to a molecule (e.g., an aptamer) that binds to a target(e.g., a protein) with at least five-fold greater affinity as comparedto any non-targets, e.g., at least 10-, 20-, 50-, or 100-fold greateraffinity.

Optional” or “optionally” means that the subsequently described event orcircumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

An “antibody” is an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term is used in the broadest sense andencompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)₂, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,fusion proteins comprising an antibody portion, and any other modifiedimmunoglobulin molecule comprising an antigen recognition site so longas the antibodies exhibit the desired biological activity. An antibodycan be of any the five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2), based on the identity of their heavy-chainconstant domains referred to as alpha, delta, epsilon, gamma, and mu,respectively. The different classes of immunoglobulins have differentand well known subunit structures and three-dimensional configurations.Antibodies can be naked or conjugated to other molecules such as toxins,radioisotopes, etc.

As used herein, the terms “antibody fragments”, “fragment”, or “fragmentthereof” refer to a portion of an intact antibody. Examples of antibodyfragments include, but are not limited to, linear antibodies;single-chain antibody molecules; Fc or Fc′ peptides, Fab and Fabfragments, and multispecific antibodies formed from antibody fragments.In most embodiments, the terms also refer to fragments that binding anantigen of a target molecule (e.g., neurogranin) and can be referred toas “antigen-binding fragments.”

As used herein, “humanized” forms of non-human (e.g., murine) antibodiesare chimeric antibodies that contain minimal sequence, or no sequence,derived from non-human immunoglobulin. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a hypervariable region of the recipient are replaced byresidues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat, rabbit or nonhuman primate having thedesired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are generally made to furtherrefine antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the hypervariable loopscorrespond to those of a nonhuman immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinsequence. The humanized antibody can also comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Examples of methods used to generate humanizedantibodies are described in U.S. Pat. No. 5,225,539.

The term “human antibody” as used herein means an antibody produced by ahuman or an antibody having an amino acid sequence corresponding to anantibody produced by a human made using any of the techniques known inthe art. This definition of a human antibody includes intact orfull-length antibodies, fragments thereof, and/or antibodies comprisingat least one human heavy and/or light chain polypeptide such as, forexample, an antibody comprising murine light chain and human heavy chainpolypeptides.

“Hybrid antibodies” are immunoglobulin molecules in which pairs of heavyand light chains from antibodies with different antigenic determinantregions are assembled together so that two different epitopes or twodifferent antigens can be recognized and bound by the resultingtetramer.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation. An antigenic determinant can compete withthe intact antigen (i.e., the “immunogen” used to elicit the immuneresponse) for binding to an antibody.

II. Neurogranin Aptamers

The present invention relates to polynucleotide aptamers thatspecifically bind to neurogranin. In certain embodiments, the aptamersare used for neurogranin detection. The sequence of the polynucleotideaptamers of the invention are disclosed herein, and further aptamerembodiments may be selected by any method known in the art. In oneembodiment, aptamers may be selected by an iterative selection processsuch as Systemic Evolution of Ligands by Exponential Enrichment (SELEX).In this type of process, a random pool of oligonucleotides (e.g., about10⁵ to about 10¹⁵ random oligonucleotides) is exposed to a targetprotein and the oligonucleotides that bind to the target are isolatedand mutagenized and the process repeated until oligonucleotides thatbind with the desired affinity to the target are identified. In anotherembodiment, aptamers may be selected by starting with the sequences andstructural requirements of the aptamers disclosed herein and modifyingthe sequences to produce other aptamers.

In one embodiment of the invention, the aptamers are directed to amammalian neurogranin protein. In further embodiments, the aptamers maybe directed to human, mouse or rat neurogranin. In another embodiment,the aptamers are directed to human, mouse and rat neurogranin. Inparticular embodiments, the aptamers may bind neurogranin with a K_(d)of less than about 1000 nM, e.g., less than about 500, 200, 100, 50, or20 nM. The aptamers may be directed to any isoform orpost-translationally modified form of neurogranin or any combination ofisoforms, post-translationally modified forms and the like.

The length of the aptamers of the invention is not limited, but typicalaptamers have a length of about 10 to about 100 nucleotides, e.g., about20 to about 80 nucleotides, about 30 to about 50 nucleotides, or about40 nucleotides. In certain embodiments, the aptamer may have additionalnucleotides attached to the 5′- and/or 3′ end. The additionalnucleotides may be, e.g., part of primer sequences, restrictionendonuclease sequences, or vector sequences useful for producing theaptamer.

The polynucleotide aptamers of the present invention may be comprisedof, ribonucleotides only (RNA aptamers), deoxyribonucleotides only (DNAaptamers), or a combination of ribonucleotides and deoxyribonucleotides.The nucleotides may be naturally occurring nucleotides (e.g., ATP, TTP,GTP, CTP, UTP) or modified nucleotides. Modified nucleotides refers tonucleotides comprising bases such as, for example, adenine, guanine,cytosine, thymine, and uracil, xanthine, inosine, and queuosine thathave been modified by the replacement or addition of one or more atomsor groups. Some examples of types of modifications that can comprisenucleotides that are modified with respect to the base moieties, includebut are not limited to, alkylated, halogenated, thiolated, aminated,amidated, or acetylated bases, in various combinations. More specificexamples include 5-propynyluridine, 5-propynylcytidine, 6-methyladenine,6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine,2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine,5-methyluridine and other nucleotides having a modification at the 5position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any O- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety(e.g., 2′-fluoro or 2′-O-methyl nucleotides), as well as nucleotideshaving sugars or analogs thereof that are not ribosyl. For example, thesugar moieties may be, or be based on, mannoses, arabinoses,glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars,heterocycles, or carbocycles. The term nucleotide is also meant toinclude what are known in the art as universal bases. By way of example,universal bases include but are not limited to 3-nitropyrrole,5-nitroindole, or nebularine. Modified nucleotides include labelednucleotides such as radioactively, enzymatically, or chromogenicallylabeled nucleotides.

In one embodiment of the invention, the aptamer is a RNA aptamer andcomprises a nucleotide sequence that is identical to any of SEQ IDNOS:4-6. In another embodiment, the RNA aptamer consists of a nucleotidesequence that is identical to any of SEQ ID NOS:4-6. In a furtherembodiment, the RNA aptamer comprises a nucleotide sequence that is atleast 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOS:4-6. Inanother embodiment, the aptamer consists of a nucleotide sequence thatis at least 70% identical, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOS:4-6.In a different embodiment, the aptamer comprises a nucleotide sequencethat is identical to a fragment of any of SEQ ID NOS:4-6 of at least 10contiguous nucleotides, e.g., at least about 15, 20, 25, 30, or 35contiguous nucleotides. In a further embodiment, the aptamer comprises anucleotide sequence that is at least 70% identical, e.g., at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%; 96%, 97%, 98%, or 99% identicalto a fragment of any of SEQ ID NOS:4-6 of at least contiguous 10nucleotides, e.g., at least about 15, 20, 25, 30, or 35 contiguousnucleotides. In one embodiment, one or more ribonucleotides in the RNAaptamers described above are substituted by a deoxyribonucleotide. Inanother embodiment, the fragments and/or analogs of the aptamers of SEQID NOS:4-6 have a substantially similar binding and/or inhibitoryactivity as one or more of the aptamers of SEQ ID NOS:4-6.“Substantially similar,” as used herein, refers to a binding and/or aninhibitory activity on one or more neurogranin functions that is atleast about 20% of the binding and/or inhibitory activity of one or moreof the aptamers of SEQ ID NOS:4-6.

Changes to the aptamer sequences, such as SEQ ID NOS:4-6, may be madebased on structural requirements for binding of the aptamers toneurogranin. The structural requirements may be readily determined byone of skill in the art by analyzing common sequences between thedisclosed aptamers and/or by mutagenizing the disclosed aptamers andmeasuring neurogranin binding affinity. For example, each of NRGN-A1,NRGN-A2, NRGN-A3, NRGN-A4 and NRGN-A5 comprise 2T-rich motifs which areseparated by CC, suggesting that this sequence is important for bindingactivity.

The aptamer may by synthesized by any method known to those of skill inthe art. In one embodiment, aptamers may be produced by chemicalsynthesis of oligonucleotides and/or ligation of shorteroligonucleotides. Another embodiment of the present invention relates topolynucleotides encoding the aptamers of the invention. Thepolynucleotides may be used to express the aptamers, e.g., by in vitrotranscription, polymerase chain reaction amplification, or cellularexpression. The polynucleotide may be DNA and/or RNA and may besingle-stranded or double-stranded. In one embodiment, thepolynucleotide is a vector which may be used to express the aptamer. Thevector may be, e.g., a plasmid vector or a viral vector and may besuited for use in any type of cell, such as mammalian, insect, plant,fungal, or bacterial cells. The vector may comprise one or moreregulatory elements necessary for expressing the aptamers, e.g., apromoter, enhancer, transcription control elements, etc. One embodimentof the invention relates to a cell comprising a polynucleotide encodingthe aptamers of the invention. In another embodiment, the inventionrelates to a cell comprising the aptamers of the invention. The cell maybe any type of cell, e.g., mammalian, insect, plant, fungal, orbacterial cells.

One aspect of the present invention relates to the use of the aptamersof the invention for diagnostic purposes. The aptamers can be used asbinding agents in assays for measuring the level of neurogranin in asubject. Such measurements can be used to determine if neurograninlevels are abnormal. Such measurements can further be used to diagnose adisease or disorder associated with neurogranin, e.g., associated withneurogranin overexpression or underexpression. In other embodiments, theaptamers can be used in neurogranin receptor competitive binding assaysto measure the abundance of neurogranin receptors and/or the bindingaffinity and specificity of neurogranin for the receptors. The aptamerscan also be used for in vivo imaging or histological analysis. Numeroussuitable binding assays are well known to those of skill in the art.Diagnostic assays can be carried out in vitro on isolated cells or celllines for research purposes. Diagnostic assays can also be carried outon samples from a subject (e.g., tissue samples (biopsies, aspirates,scrapings, etc.) or body fluid samples (blood, plasma, serum, saliva,urine, cerebrospinal fluid, etc.)) or carried out in vivo. The aptamerscan be labeled using methods and labels known in the art including, butnot limited to, fluorescent, luminescent, phosphorescent, radioactive,and/or colorimetric compounds.

In one aspect, the invention relates to a method of measuring the levelof neurogranin in a subject, comprising the step of using thepolynucleotide aptamer of the invention to bind neurogranin. In anotheraspect, the invention relates to a method of diagnosing a disease ordisorder associated with neurogranin in a subject, comprising the stepof measuring the level of neurogranin in the subject using thepolynucleotide aptamer of the invention. The level of neurogranin canthen be correlated with the presence or absence of a disease or disorderassociated with neurogranin.

For each of the methods described above, the methods may be carried outusing a single aptamer targeted to neurogranin. In another embodiment,the methods may be carried out using two or more different aptamerstargeted to neurogranin, e.g., three, four, five, or six differentaptamers.

III. Neurogranin Antibodies

In one aspect, the present invention provides antibodies to neurograninthat are useful for diagnostic or screening purposes. In certainembodiments, the antibodies described herein are isolated. In certainembodiments, the antibodies described herein are substantially pure.

In some embodiments the antibodies are monoclonal antibodies. In certainembodiments, the antibodies are chimeric, humanized, or humanantibodies. The invention further provides bispecific antibodies. Incertain embodiments, the antibodies are antibody fragments, such as Fabfragments.

In particular embodiments, the present invention provides isolatedantibodies against neurogranin. In a specific embodiment, the antibodiesare specific for SEQ ID NO:11. In other embodiments, the antibodiesspecifically bind amino acids 55-78 of SEQ ID NO:11. The antibody, orantibody fragment thereof, can be any monoclonal or polyclonal antibodythat specifically recognizes neurogranin. In some embodiments, thepresent invention provides monoclonal antibodies, or fragments thereof,that specifically bind to neurogranin. In some embodiments, themonoclonal antibodies, or fragments thereof, are chimeric or humanizedantibodies that specifically bind to neurogranin or an eptiope orantigenic determinant thereof.

The antibodies against neurogranin find use in the experimental anddiagnostic methods described herein. In certain embodiments, theantibodies of the present invention are used to detect the expression ofa neurogranin protein in biological samples such as, for example, atissue, blood, plasma, serum, cerebrospinal fluid sample and the like.Tissue biopsies can be sectioned and neurogranin protein detected using,for example, immunofluorescence or immunohistochemistry. Alternatively,individual cells from a sample are isolated, and protein expressiondetected on fixed or live cells by FACS analysis. Furthermore, theantibodies can be used on protein arrays to detect expression ofneurogranin, for example, on cells, in cell lysates, or in other proteinsamples.

Polyclonal antibodies can be prepared by any known method. Polyclonalantibodies can be raised by immunizing an animal (e.g., a rabbit, rat,mouse, donkey, etc) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (a purified peptide fragment,full-length recombinant protein, fusion protein, etc) optionallyconjugated to keyhole limpet hemocyanin (KLH), serum albumin, etc.diluted in sterile saline and combined with an adjuvant (e.g., Completeor Incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized. Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Alternatively, lymphocytes can be immunized in vitro. Followingimmunization, the lymphocytes are isolated and fused with a suitablemyeloma cell line using, for example, polyethylene glycol, to formhybridoma cells that can then be selected away from unfused lymphocytesand myeloma cells. Hybridomas that produce monoclonal antibodiesdirected specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assaysuch as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA) can then be propagated either in vitro culture using standardmethods (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, 1986) or in vivo as ascites tumors in an animal. Themonoclonal antibodies can then be purified from the culture medium orascites fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated, such as from mature B-cellsor hybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries as described (McCafferty et al., 1990, Nature,348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks etal., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different ways using recombinant DNA technologyto generate alternative antibodies. In one embodiment, the constantdomains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In otherembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Furthermore,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of a monoclonal antibody.

In some embodiments, of the present invention the monoclonal antibodyagainst neurogranin is a humanized antibody. Humanized antibodies areantibodies that contain minimal sequences from non-human (e.g., murine)antibodies within the variable regions. In practice, humanizedantibodies are typically human antibodies with minimum to no non-humansequences. A human antibody is an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDR of a humanantibody with that of a non-human antibody (e.g., mouse, rat, rabbit,hamster, etc.) having the desired specificity, affinity, and capability(Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988,Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536).The humanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, for example, Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat.No. 5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies (Vaughan etal., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS,95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Markset al., 1991, J. Mol. Biol., 222:581). Humanized antibodies can also bemade in transgenic mice containing human immunoglobulin loci that arecapable upon immunization of producing the full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Thisapproach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and 5,661,016.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody. Various techniquesare known for the production of antibody fragments. Traditionally, thesefragments are derived via proteolytic digestion of intact antibodies(for example Morimoto et al., 1993, Journal of Biochemical andBiophysical Methods 24:107-117 and Brennan et al., 1985, Science,229:81). However, these fragments are now typically produced directly byrecombinant host cells as described above. Thus Fab, Fv, and scFvantibody fragments can all be expressed in and secreted from E. coli orother host cells, thus allowing the production of large amounts of thesefragments. Alternatively, such antibody fragments can be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e., thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The invention further provides kits and articles of manufacturecomprising one or more antibodies. In certain embodiments, the kitscomprise at least two antibodies. In certain embodiments, the kitscomprise at least one antibody that specifically binds a neurograninprotein.

IV. Detection of Neurogranin and Other Biomarkers

A. Detection by Mass Spectrometry

In one aspect, the biomarkers of the present invention may be detectedby mass spectrometry, a method that employs a mass spectrometer todetect gas phase ions. Examples of mass spectrometers aretime-of-flight, magnetic sector, quadrupole filter, ion trap, ioncyclotron resonance, electrostatic sector analyzer, hybrids orcombinations of the foregoing, and the like. In a specific embodiment,the mass spectrometric method comprises matrix assisted laserdesorption/ionization time-of-flight (MALDI-TOF MS or MALDI-TOF). Inanother embodiment, method comprises MALDI-TOF tandem mass spectrometry(MALDI-TOF MS/MS). In yet another embodiment, mass spectrometry can becombined with another appropriate method(s) as may be contemplated byone of ordinary skill in the art. For example, MALDI-TOF can be utilizedwith trypsin digestion and tandem mass spectrometry as described herein.In another embodiment, the mass spectrometric technique is multiplereaction monitoring (MRM) or quantitative MRM.

In an alternative embodiment, the mass spectrometric technique comprisessurface enhanced laser desorption and ionization or “SELDI,” asdescribed, for example, in U.S. Pat. No. 6,225,047 and No. 5,719,060.Briefly, SELDI refers to a method of desorption/ionization gas phase ionspectrometry (e.g. mass spectrometry) in which an analyte (here, one ormore of the biomarkers) is captured on the surface of a SELDI massspectrometry probe. There are several versions of SELDI that may beutilized including, but not limited to, Affinity Capture MassSpectrometry (also called Surface-Enhanced Affinity Capture (SEAC)), andSurface-Enhanced Neat Desorption (SEND) which involves the use of probescomprising energy absorbing molecules that are chemically bound to theprobe surface (SEND probe). Another SELDI method is calledSurface-Enhanced Photolabile Attachment and Release (SEPAR), whichinvolves the use of probes having moieties attached to the surface thatcan covalently bind an analyte, and then release the analyte throughbreaking a photolabile bond in the moiety after exposure to light, e.g.,to laser light (see, U.S. Pat. No. 5,719,060). SEPAR and other forms ofSELDI are readily adapted to detecting a biomarker or biomarker panel,pursuant to the present invention.

In another mass spectrometry method, the biomarkers can be firstcaptured on a chromatographic resin having chromatographic propertiesthat bind the biomarkers. For example, one could capture the biomarkerson a cation exchange resin, such as CM Ceramic HyperD F resin, wash theresin, elute the biomarkers and detect by MALDI. Alternatively, thismethod could be preceded by fractionating the sample on an anionexchange resin before application to the cation exchange resin. Inanother alternative, one could fractionate on an anion exchange resinand detect by MALDI directly. In yet another method, one could capturethe biomarkers on an immuno-chromatographic resin that comprisesantibodies that bind the biomarkers, wash the resin to remove unboundmaterial, elute the biomarkers from the resin and detect the elutedbiomarkers by MALDI or by SELDI.

B. Detection by Immunoassay

In other embodiments, the biomarkers of the present invention can bedetected and/or measured by immunoassay Immunoassay requires biospecificcapture reagents, such as antibodies, to capture the biomarkers. Manyantibodies are available commercially. Antibodies also can be producedby methods well known in the art, e.g., by immunizing animals with thebiomarkers. Biomarkers can be isolated from samples based on theirbinding characteristics. Alternatively, if the amino acid sequence of apolypeptide biomarker is known, the polypeptide can be synthesized andused to generate antibodies by methods well-known in the art.

The present invention contemplates traditional immunoassays including,for example, sandwich immunoassays including ELISA or fluorescence-basedimmunoassays, immunoblots, Western Blots (WB), as well as other enzymeimmunoassays. Nephelometry is an assay performed in liquid phase, inwhich antibodies are in solution. Binding of the antigen to the antibodyresults in changes in absorbance, which is measured. In a SELDI-basedimmunoassay, a biospecific capture reagent for the biomarker is attachedto the surface of an MS probe, such as a pre-activated protein chiparray. The biomarker is then specifically captured on the biochipthrough this reagent, and the captured biomarker is detected by massspectrometry.

Although antibodies are useful because of their extensivecharacterization, any other suitable agent (e.g., a peptide, an aptamer,or a small organic molecule) that specifically binds a biomarker of thepresent invention is optionally used in place of the antibody in theabove described immunoassays. For example, an aptamer that specificallybinds all neurogranin and/or one or more of its breakdown products mightbe used. Aptamers are nucleic acid-based molecules that bind specificligands. Methods for making aptamers with a particular bindingspecificity are known as detailed in U.S. Pat. No. 5,475,096; U.S. Pat.No. 5,670,637; U.S. Pat. No. 5,696,249; U.S. Pat. No. 5,270,163; U.S.Pat. No. 5,707,796; U.S. Pat. No. 5,595,877; U.S. Pat. No. 5,660,985;U.S. Pat. No. 5,567,588; U.S. Pat. No. 5,683,867; U.S. Pat. No.5,637,459; and U.S. Pat. No. 6,011,020.

C. Detection by Electrochemicaluminescent Assay

In several embodiments, the biomarker biomarkers of the presentinvention may be detected by means of an electrochemicaluminescent assaydeveloped by Meso Scale Discovery (Gaithersrburg, Md.).Electrochemiluminescence detection uses labels that emit light whenelectrochemically stimulated. Background signals are minimal because thestimulation mechanism (electricity) is decoupled from the signal(light). Labels are stable, non-radioactive and offer a choice ofconvenient coupling chemistries. They emit light at ˜620 nm, eliminatingproblems with color quenching. See U.S. Pat. No. 7,497,997; U.S. Pat.No. 7,491,540; U.S. Pat. No. 7,288,410; U.S. Pat. No. 7,036,946; U.S.Pat. No. 7,052,861; U.S. Pat. No. 6,977,722; U.S. Pat. No. 6,919,173;U.S. Pat. No. 6,673,533; U.S. Pat. No. 6,413,783; U.S. Pat. No.6,362,011; U.S. Pat. No. 6,319,670; U.S. Pat. No. 6,207,369; U.S. Pat.No. 6,140,045; U.S. Pat. No. 6,090,545; and U.S. Pat. No. 5,866,434. Seealso U.S. Patent Applications Publication No. 2009/0170121; No.2009/006339; No. 2009/0065357; No. 2006/0172340; No. 2006/0019319; No.2005/0142033; No. 2005/0052646; No. 2004/0022677; No. 2003/0124572; No.2003/0113713; No. 2003/0003460; No. 2002/0137234; No. 2002/0086335; andNo. 2001/0021534.

D. Other Methods for Detecting Biomarkers

The biomarkers of the present invention can be detected by othersuitable methods. Detection paradigms that can be employed to this endinclude optical methods, electrochemical methods (voltametry andamperometry techniques), atomic force microscopy, and radio frequencymethods, e.g., multipolar resonance spectroscopy. Illustrative ofoptical methods, in addition to microscopy, both confocal andnon-confocal, are detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, andbirefringence or refractive index (e.g., surface plasmon resonance,ellipsometry, a resonant mirror method, a grating coupler waveguidemethod or interferometry).

Furthermore, a sample may also be analyzed by means of a biochip.Biochips generally comprise solid substrates and have a generally planarsurface, to which a capture reagent (also called an adsorbent oraffinity reagent) is attached. Frequently, the surface of a biochipcomprises a plurality of addressable locations, each of which has thecapture reagent bound there. Protein biochips are biochips adapted forthe capture of polypeptides. Many protein biochips are described in theart. These include, for example, protein biochips produced by CiphergenBiosystems, Inc. (Fremont, Calif.), Invitrogen Corp. (Carlsbad, Calif.),Affymetrix, Inc. (Fremong, Calif.), Zyomyx (Hayward, Calif.), R&DSystems, Inc. (Minneapolis, Minn.), Biacore (Uppsala, Sweden) andProcognia (Berkshire, UK). Examples of such protein biochips aredescribed in the following patents or published patent applications:U.S. Pat. No. 6,537,749; U.S. Pat. No. 6,329,209; U.S. Pat. No.6,225,047; U.S. Pat. No. 5,242,828; PCT International Publication No. WO00/56934; and PCT International Publication No. WO 03/048768.

V. Kits for the Detection of Neurogranin and Other Brain InjuryBiomarkers

In another aspect, the present invention provides kits for qualifyingbrain injury status, which kits are used to detect neurogranin and otherbiomarkers. In a specific embodiment, the kit is provided as an ELISAkit comprising antibodies to neurogranin. In other embodiments, a kitcan comprise antibodies to one or more of ASTN1, BAI3, CNDP1, ERMIN,GFAP, GRM3, KLH32, MAGE2, NRG3, OMG, SLC39A12, RTN1, and MT3.

The ELISA kit may comprise a solid support, such as a chip, microtiterplate (e.g., a 96-well plate), bead, or resin having biomarker capturereagents attached thereon. The kit may further comprise a means fordetecting the biomarkers, such as antibodies, and a secondaryantibody-signal complex such as horseradish peroxidase (HRP)-conjugatedgoat anti-rabbit IgG antibody and tetramethyl benzidine (TMB) as asubstrate for HRP.

In other embodiments, the kit for qualifying brain injury status may beprovided as an immuno-chromatography strip comprising a membrane onwhich the antibodies are immobilized, and a means for detecting, e.g.,gold particle bound antibodies, where the membrane, includes NC membraneand PVDF membrane. The kit may comprise a plastic plate on which asample application pad, gold particle bound antibodies temporallyimmobilized on a glass fiber filter, a nitrocellulose membrane on whichantibody bands and a secondary antibody band are immobilized and anabsorbent pad are positioned in a serial manner, so as to keepcontinuous capillary flow of blood serum.

In certain embodiments, a patient can be diagnosed by adding blood orblood serum from the patient to the kit and detecting the relevantbiomarkers conjugated with antibodies, specifically, by a method whichcomprises the steps of: (i) collecting blood or blood serum from thepatient; (ii) separating blood serum from the patient's blood; (iii)adding the blood serum from patient to a diagnostic kit; and, (iv)detecting the biomarkers conjugated with antibodies. In this method, theantibodies are brought into contact with the patient's blood. If thebiomarkers are present in the sample, the antibodies will bind to thesample, or a portion thereof. In other kit and diagnostic embodiments,blood or blood serum need not be collected from the patient (i.e., it isalready collected). Moreover, in other embodiments, the sample maycomprise a tissue sample or a clinical sample.

The kit can also comprise a washing solution or instructions for makinga washing solution, in which the combination of the capture reagents andthe washing solution allows capture of the biomarkers on the solidsupport for subsequent detection by, e.g., antibodies or massspectrometry. In a further embodiment, a kit can comprise instructionsfor suitable operational parameters in the form of a label or separateinsert. For example, the instructions may inform a consumer about how tocollect the sample, how to wash the probe or the particular biomarkersto be detected, etc. In yet another embodiment, the kit can comprise oneor more containers with biomarker samples, to be used as standard(s) forcalibration.

Without further elaboration, it is believed that one skilled in the art,using the preceding description, can utilize the present invention tothe fullest extent. The following examples are illustrative only, andnot limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices, and/or methods described andclaimed herein are made and evaluated, and are intended to be purelyillustrative and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.) butsome errors and deviations should be accounted for herein. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees Celsius or is at ambient temperature, and pressure is at or nearatmospheric. There are numerous variations and combinations of reactionconditions, e.g., component concentrations, desired solvents, solventmixtures, temperatures, pressures and other reaction ranges andconditions that can be used to optimize the product purity and yieldobtained from the described process. Only reasonable and routineexperimentation will be required to optimize such process conditions.

Example 1 Development of Neurogranin Assay

The Identification of Human NRGN Specific Aptamers.

Systematic Evolution of Ligands by EXponential enrichment (SELEX)procedure was used to identify the human NRGN specific aptamers.Briefly, the specific aptamer was selected from a pool of single strandRNA by filter immobilization. The RNA-NRGN target complex can bind tonitrocellulose filter, and free RNA went through filtration. Thespecific aptamer was recovered from the filter and PCR amplified. Afterseveral round of selection, the specific aptamer with highest affinitywith NRGN was enriched and sequencing identified.

Prepare the Library for Selection.

Single strand DNA oligo pool was chemically synthesized. The DNA oligohas 40mer random central core, which flanked by 2 constant sequences,and the library can be amplified by a pair of primers which target the5′ and 3′ conserve ends of the library. The sequence of the library is5′-TCTCGGATCCTCAGCGAG TCGTCTG (N40) CCGCATCGTCCTCCCTA-3′ (SEQ ID NO:1).

Generating RNA Library.

The single strand DNA library first was annealed with Sel2 5′ primer,the sequence is 5′-GGGGGAATTCTAATACGACTCACTATAGGGAGGACG ATGCGG-3′ (SEQID NO:2), which contains a T7 promoter for in vitro transcription. Thegap was filled in by Klenow reaction, which was performed at 37° C. for1.5 hours. The reaction was purified by phenol:chloroform:isoamyl(25:24:1) and chloroform:isoamyl (24:1) extraction once each and furtherconcentrated with Centricon 30 at 4° C. TE buffer, pH7.4 was used towash the reaction twice while concentration process. The final OD260 wasmeasured and concentration was determined.

The above annealed oligo pool was used to generate RNA library for SELEXby in vitro transcription, using the DuraScribe® T7 Transcription Kits(Epicentre, DS010910), following the manufacture's reaction condition.2′-Fluorine-CTP (2′-F-dCTP) and 2′-Fluorine-UTP (2′-F-dUTP) were used toreplace CTP and UTP in the transcription reaction, the final DuraScript®RNA (2′-fluorine-modified RNA) is completely resistant to RNase A. Afterin vitro transcription, DNase I was used to treat the reaction and theRNA was extracted with phenol:chloroform:isoamyl (25:24:1) andchloroform once each, followed by concentrate and desalt with Centricon30 at 4° C.

The RNA was further purified by denatured PAGE (12%, 7M Urea) gelpurification. The RNA band was cut from the PAGE gel, RNA was eluted in2 ml TE buffer overnight at 4° C. The pure RNA was concentrate againusing Centricon 30, and concentration of RNA was determined usingconventional method.

Nitrocellulose Filter Pre-Clear.

A 13 mm Swin-Lok Filter holder (Whatman) (13-mm diameter), 0.45 um poresize HAWP nitrocellulose disk filters (Millipore) was assembled. Thefilter was pre-wet with 1 ml washing buffer, which contains 20 mM HepespH7.4, 50 mM NaCl and 2 mM CaCl₂. 500 pmol RNA was diluted in 100 ul 1×binding buffer (the formula is the same as washing buffer except 0.1%BSA was added), and applied into the reservoir of the filter holder. Thefilter holder was sealed into a 50 ml conical tube and incubated at 37°C. for 30 minutes. After incubation, the RNA was recovered by passthrough the filter unite using 1 ml syringe, and 100 μl of 1× bindingbuffer was used to wash once. The RNA passed through the filter wascollected; total pre-cleared RNA was about 180 μl.

Binding Reaction.

The binding reaction was assembled by adding 50 pmol human NRGN proteininto the pre-cleared RNA, the molecular ratio of RNA:protein was about10:1. The total volume was brought to 200 μl in 1× binding buffer. Thereaction was incubated at 37° C. for 15 minutes. A new filter holder wasassembled and prewet as above, the binding reaction to the filter wasapplied, a 5 ml syringe was used to push the binding sample through, andthe filter was washed with 5 ml wash buffer.

Recover the Selected RNA.

The filter holder was disassembled, and the filter was transferred intoa 1.5 ml centrifuge tube which contained 600 ulphenol:chloroform:isoamyl (25:24:1). The tube was vortexed vigorouslyfor approximately 1 min, then incubated at RT for 30 minutes. Twohundred microliters of H₂O was added and vortexed again, then spun at14,000 rpm for 10 minutes. The supernatant, which contained therecovered RNA, was extracted with 400 ul chloroform once, thenprecipitated by adding 500 μl ethanol, 20 μl 3M NaAcetate (pH 5.2) and 3μl Glycogen blue (Ambion, 5 mg/ml), and then incubated at −80° C.overnight. The RNA was recovered by centrifugation at 14000 rpm for 20minutes at 4° C., then washed with 1 ml 75% ethanol once, followed bycentrifugation and air drying of the RNA. The dried RNA pellet wasdissolved into 20 μl H₂O.

Amplify the Selected RNA by RT-PCR.

Five microliters of recovered RNA was used to synthesize the firststrand of DNA. Two micromolar of primer Sel2 3′ was added into thereaction. The sequence of Sel2 3′ primer is: 5′-TCT CGG ATC CTC AGC GAGTCG TC-3′ (SEQ ID NO:3). Reverse Transcriptase from Roche (Cat. No. 10109 118 001) was used in the reaction, the conditions were asrecommended by the manufacture.

The PCR reaction was assembled as follows: 5 μl first stand DNA (fromabove step), 3 μl of each 10 μM primers (Sel2 3′ from above step andSel2 5′ from above step), 39 μl H2O and 50 μl 2× TopTaq Master Mix(Qiagen). A total of 8 reactions (800 ul) were performed. The PCR cyclecondition was as follow: 94° C./5′->(94° C./30″->55° C./30″->72°C./30″)×20 cycles->4° C. The PCR product was confirmed by 3% agarose gelelectrophoresis, and the rest of PCR product were desalted andconcentrated using a Centricon 30 at 4° C. The concentration of PCRproduct was determined by measuring OD/260 nm.

Repeat the Selection.

One microgram of concentrated PCR product was used to generate RNA forthe next round selection, the protocols for in vitro transcription werefollowed as described above. A total of 10 rounds of selection wasperformed.

After 10 rounds of selection, another 3 rounds of selection wereperformed using high-salt binding buffer to increase the selectionstringency. The formula for 1× binding buffer F and washing buffer F isthe same as the buffers described above, except that the concentrationof NaCl was increased to 150 mM. The other detailed procedure is thesame as that described above.

The final PCR product after 13 rounds of selection was cloned intopGEM-T Easy vector (Promega), the enriched aptamers were identified byDNA sequencing. The clones containing the full primers and 40mer insertwere aligned using ClustalW2 at EMBL-EBI website.

The Identified Human NRGN Specific Aptamers.

Six clones were chosen for sequencing and all the sequences' qualitywere very high. After the DNA alignment analysis, 5 out of the 6 cloneswere almost identical, except that NRGN-A4 had one nucleotide difference(underlined). The less similar one, NRGN-A6, comparing with the otherwell aligned 5 clones, showed that all of them have 2 T-rich motifs,which are separated by CC/CA (boxed). NRGN-A1 and NRGN-A6 were selectedas targets for validation. The following showed the alignment of theaptamers:

(SEQ ID NO: 4)

(SEQ ID NO: 4)

(SEQ ID NO: 4)

(SEQ ID NO: 4)

(SEQ ID NO: 5)

(SEQ ID NO: 6)

Validation of the Aptamer-NRGN Interaction.

NRGN-A1 and NRGN-A6 RNA were chemically synthesized based on thesequences identified; adding a biotin linker to the RNA 3′ end. Twodifferent strategies were used to test NRGN aptamer and NRGN recombinantprotein interaction. The details are as described below.

Using Dot Blot to Detect the RNA Protein Complex on Nitrocellulose.

Based on the same mechanism that was used in SELEX procedure,RNA-protein complex can be retained on nitrocellulose membrane, a dotblot was used to detect the biotin labeled RNA aptamer. First, a 2-foldseries dilution of His-NRGN recombinant protein was made, the amount ofprotein range from 10 pmol to 0; and then each sample was mixed with 1pmol of NRGN aptamer RNA. The volume of final binding reaction was keptat 20 μl in 1× binding buffer F, the reactions were incubated at 37° C.for 15 min.

Meanwhile, the dot blot apparatus (Bio-Rad, #170-6545) was setup. Thenitrocellulose membrane was cut and shaken in 1× washing buffer F for 30min prior to use. The nitrocellulose membrane was put on top of pre-wetWhatman paper and placed on the bottom of the apparatus, then the vacuumwas assembled and hooked up. The membrane was washed with 100 ul 1×washing buffer F per well once, then the binding reaction was applied.After the reactions were passed through, the membrane was washed with200 ul 1× washing buffer F once, and then drained by vacuuming. Themembrane was then UV-crosslinked (Bio-Rad, #165-5031).

The Chemiluminescent Nucleic Acid Detection Module (Pierce, #89880) wasused to detect the Biotin labeled RNA aptamer retained onnitrocellulose, following the manufacturer's instruction. Briefly, themembrane was incubated with streptavidin-HRP conjugate, and detectedwith the chemiluminescent substrate of HRP.

As a result, both NRGN aptamers NRGN-A1 and A6 bound to NRGN protein,the minimum amount of protein needed for detection using this method was1.25 pmol and 0.63 pmol respectfully. When the same molar of humanalbumin was used as control, no signal could be detected, despite theamount of albumin used. This result indicated that both of these 2aptamers bind to His-NRGN protein specifically. See FIG. 1.

Pull-Down Assay.

Biotin labeled aptamers were immobilized on streptavidin particles and apull-down assay was performed. Aptamers were diluted to 1 pmol/μl inTEN100 buffer (10 mM Tris-HCl, pH7.5, 1 mM EDTA, 100 mM NaCl), heated at65° C. for 5′, then left at RT for 20 minutes to let the RNA fold intoits natural conformation. Streptavidin magnetic particles (Roche,11641778001) were washed 3 times with twice volume of TEN100 buffer,then the aptamer samples were added and incubated at RT for 30 minutewith rotation. After incubation, the particles were washed with TEN100buffer 3 times, and then equilibrated with 1× binding buffer F once.Different amounts of NRGN protein (0-2 nmol) were diluted in 1× bindingbuffer and added into the aptamer immobilized magnetic particles, thenincubated at 37° C. for 15 minutes.

After binding incubation, the particles were washed twice with TEN100buffer. The aptamers protein complex was dissociated by incubating in 26μl elution buffer (1M NaCl, 10 mM EDTA) at room temperature for 10 min.The eluted protein was subjected to SDS-PAGE, and the protein bands werevisualized by coomassie staining. The human albumin protein was used asnegative control. A typical stained gel is shown in FIG. 2.

Both of the dot blot and pull-down assays showed that the aptamersspecifically bind to human NRGN recombinant protein.

Development of a Neurogranin Multiple Reaction Monitoring (MRM) Assay.

A neurogranin signature peptide was developed for a mass spectroscopyquantitative MRM assay. The peptide sequence and transitions are shownin the table below. Labeled GPGPGGPGGAGVAR (SEQ ID NO:7) was spiked inthe samples to make standard curve to measure the concentration ofsignature peptide GPGPGGPGGAGVAR (SEQ ID NO:7). Peptide KGPGPGGPGGAGVAR(SEQ ID NO:8) is also monitored to make sure there is no miscleavage intryptic digestion.

TABLE 8 Neurogranin Peptide Sequences Precursor Transitions Q1 Q3Peptide ID 553.79 366.18 GPGPGGPGGAGVAR  (SEQ ID NO: 7) 553.79 423.2GPGPGGPGGAGVAR  (SEQ ID NO: 7) 553.79 684.38 GPGPGGPGGAGVAR (SEQ ID NO: 7) 553.79 741.4 GPGPGGPGGAGVAR  (SEQ ID NO: 7) 553.79 798.42GPGPGGPGGAGVAR  (SEQ ID NO: 7) 553.79 895.47 GPGPGGPGGAGVAR (SEQ ID NO: 7) 553.79 952.49 GPGPGGPGGAGVAR  (SEQ ID NO: 7) 558.792366.18 Labeled GPGPGGPGGAGVAR  (SEQ ID NO: 7) 558.792 423.2Labeled GPGPGGPGGAGVAR  (SEQ ID NO: 7) 558.792 694.39Labeled GPGPGGPGGAGVAR  (SEQ ID NO: 7) 558.792 751.41Labeled GPGPGGPGGAGVAR  (SEQ ID NO: 7) 558.792 808.43Labeled GPGPGGPGGAGVAR  (SEQ ID NO: 7) 558.792 905.48Labeled GPGPGGPGGAGVAR  (SEQ ID NO: 7) 617.846 684.38 KGPGPGGPGGAGVAR (SEQ ID NO: 8) 617.846 741.4 KGPGPGGPGGAGVAR  (SEQ ID NO: 8) 617.846798.42 KGPGPGGPGGAGVAR  (SEQ ID NO: 8) 617.846 950.5 KGPGPGGPGGAGVAR (SEQ ID NO: 8) 617.846 962.5 KGPGPGGPGGAGVAR  (SEQ ID NO: 8)

The signals of neurogranin signature peptide and labeled standardpeptide using an ABI Sciex Qtrap 4000 triple quadrapole massspectrometer are shown in FIG. 3.

His-NRGN Recombinant Protein Production.

Human NRGN cDNA clone was purchased from Origene (Cat. No. RC201209).The coding sequence was cloned into destination vector (Origene,pEX-N-His, Cat. No. PS100030) by restriction enzymes (Sgf I+Mlu I)fragment swapping to generate pEX-N-His-NRGN expression plasmid. Thecoding sequence and reading frame were confirmed by DNA sequencing.

pEX-N-His-NRGN plasmid was transformed into Rosetta 2 (DE3) competentcells (Novagen #71397) according to manufacturer's instruction. Thebacteria were cultured in the Overnight Express Instant TB Medium(Novagen #71491) at 37° C. for 16-18 hours, then harvested and suspendedin TEN buffer (50 mM Tris, pH8.0, 0.5 mM EDTA and 0.5 M NaCl). Thebacteria were lysated by adding 1% NP-40, 25 mg lysozyme and completeproteinase inhibitors (Roche), sitting on ice for 30 minutes, thenfreeze-thaw one time. The lysate was cleared by centrifugation, thenNi-NTA agarose beads (Qiagen) were added into the supernatant, androtated at 4° C. for 1 hour. The beads were washed 3 times with washingbuffer (20 mM Imidazole, 20 mM KCl and 0.5 M NaCl). The recombinantprotein was eluted off the beads by rotating the beads in the elutionbuffer (100 mM Imidazole, 20 mM K₃PO₄ and 167 mM NaCl) at 4° C. for 10minutes. The eluted protein was dialyzed against 3 L PBS overnight, theprotein concentration was determined by conventional protein assay. FIG.4 shows the typical His-NRGN on PAGE gel after coomassie staining; thepredicted molecular weight of His-NRGN is 8.5 Kd.

Development of a Neurogranin Monoclonal.

Recombinant neurogranin described above was used to immunize mice formonoclonal antibody production at Johns Hopkins. Thirty clones werescreened and clone 30.5.2 was identified that bound neurogranin at highdilution in a direct ELISA shown in FIG. 5.

Development of a Sandwich ELISA for Neurogranin.

A Neurogranin anti-Human monoclonal antibody (Johns Hopkins) atconcentration of 75 ng/well was used as a capture antibody and a rabbitpolyclonal to neurogranin (Johns Hopkins) at a concentration of 0.5μg/ml was used as detection. SULFO-TAG anti rabbit antibody (MSDCat#R32AB-1) at a concentration of 1 μg/ml was used as a labeledreporter at a concentration of 1 μg/ml. GST_NRGN recombinant protein(Johns Hopkins) was used as a standard at a starting concentration of 20ng/ml then at 1:2 for 7 dilutions in PBS/1% BSA. PBS/1% BSA was used asblank. The standard curve for this assay is shown in FIG. 6.

Neurogranin is Biomarker of Acute Brain Injury.

Using the neurogranin sandwich assay described above, serum samples froman infant on ECMO support for 27 days for cardio-respiratory failure.The infant had normal daily head ultrasounds and at the time of deathwas thought to not have brain injury. At autopsy, the brain had multiplecortical infarcts they were not diagnosed by ultrasound. As shown inFIG. 8, GFAP levels were unchanged during the entire coarse of ECMOsupport. However, neurogranin levels increased to a peak 15 fold overbaseline over 14 days of ECMO support. As neurogranin is a gray matter,neuronal marker it was more sensitive to cortical gray matter injurythan GFAP a marker of white matter injury. This provides evidence thatneurogranin is a circulating biomarker of acute cortical brain injuryand in combination with GFAP is able to discrimate white matter fromgray matter injury to the brain.

Example 2 Proteomic Study to Identify Brain Proteins Reveals Elevationsof Neurogranin in Children with Sickle Cell Disease

Silent cerebral infarct (SCI) is the most common form of neurologicinjury in sickle cell disease. It is associated with decreasedneurocognitive function, and increased risk for progressive injury,including stroke and new or progressive lesions. SCI is defined as anyischemic lesion visible on multiple T2-weighted magnetic resonanceimages (MRI) that is not associated with a history or physical examsuggestive of a focal neurologic deficit. To date, a noninvasive,unbiased laboratory test for SCI does not exist, and the ability toidentify children with SCD who are at risk for SCI remains limited.Furthermore, the diagnosis of SCI is made with surveillance MRI, whichis costly and not done routinely at many institutions. As a resultpatients are often diagnosed after they have experienced SCI-relatedimpairments.

The identification of plasma brain proteins that can be used asbiomarkers of SCI would provide important progress in detection andtherapy for children with SCD. Specifically, these potential biomarkerswould aid in the identification of children who are at risk for SCI,provide a cost-effective alternative to MRI for early diagnosis, andwould allow for monitoring response to treatment. Importantly,biomarkers of SCI would provide insight into the pathological mechanismsinvolved in the disease. Proteomics methods based on mass spectrometry(MS) provide a platform for the identification, quantification andcharacterization of these potential biomarkers.

In fact, proteomics has been used for biomarker discovery of brainproteins in a number of disease states, including brain cancer,alzheimer's disease, traumatic brain injury, and stroke. Massspectrometry-based approaches have also been used to gain insight intothe pathophysiology of SCD. However, very few studies have used plasmaproteomics for clinical biomarker discovery in SCD, and none have beenpublished about SCD and subclinical brain injury. Kakhniashvili et alused two-dimensional fluorescence difference gel electrophoresis (2DDIGE) and tandem MS (LC-MS/MS) to evaluate quantitative changes in thered blood cell (RBC) membrane proteome and reported on elevations ofproteins involved in repair after oxidative stress. Others have usedsurface-enhanced laser desorption/ionization time of flight (SELDI-TOF)and Matrix-assisted laser desorption/ionization (MALDI)-TOF MS toevaluate for biomarkers of pulmonary hypertension and acute painfulepisodes in SCD. The purpose of this study was to use proteomics toidentify and validate plasma brain proteins in children with SCD. Thepresent inventors hypothesized that children with SCD and SCI would havecirculating plasma brain proteins that could be used as surrogatemarkers for brain injury, and provide insight into the pathophysiologyof the disease.

Materials and Methods

Study Population.

The study population was comprised of three groups: children with SCDand SCI, children with SCD and no SCI, and healthy, age-matched controlswith no SCD or SCI. Proteomic analysis was done using plasma from sevenchildren with SCD and SCI and six children with SCD but without SCI whowere matched for age, hemoglobin and WBC based on current knowledge ofrisk factors for SCI, as well as on six age-matched African Americancontrols (three with sickle cell trait [SCT]). Cross-sectional plasmasamples from a total of 115 children with SCD (64 with SCI and 51without SCI) and 46 age-matched, African-American controls were used tomeasure plasma concentrations of identified proteins.

Plasma from children with SCD was randomly selected from those enrolledin the Silent Infarct Transfusion (SIT) Trial (ClinicalTrials.govidentifier NCT00072761). The SIT trial is a multicenter, internationalclinical study to determine if chronic blood transfusions caneffectively prevent the progression of SCI to stroke, in children withSCD aged 5-14 years. Plasma samples and clinical data for healthycontrols, without evidence of acute or chronic illness (excludingasthma, behavior/mood disorders, and obesity), were obtained through twoseparate Institutional Review Board-approved studies.

Sample Preparation.

Baseline steady state peripheral whole blood obtained from children withSCD was collected in EDTA vacutainer tubes (BD, Franklin Lakes, N.J.) atthe time of initial screening, and was shipped at room temperaturewithin 24 hours to the SIT Trial Biologic Repository at Johns Hopkins.Upon arrival, samples were and spun at 1500 g for 8 minutes perprotocol; plasma was removed and stored at −80° C. until assayed. Steadystate peripheral whole blood for the non-SCD controls used for proteomicanalysis was collected in EDTA vacutainer tubes, centrifuged at 1500 g(4° C.) for 12 minutes, then aliquoted and stored at −80° C. Plasmasamples for the healthy controls used to measure candidate proteins werealso collected in EDTA vacutainer tubes and processed according to theSIT Trial protocol.

Hemoglobin Depletion.

Significant hemolysis was observed in the discovery SCD samples. Toenrich for low abundance proteins, hemoglobin was depleted from SCDplasma samples (n=15) using nickel-nitrilotriacetic acid (Ni-NTA) beads(Qiagen). Briefly, after Ni-NTA beads were washed with PBS/0.3 M NaCL,500 μl of plasma was added to 500 μl of 50% nickel beads (250 μl beadsin 250 μl PBS/0.3 M NaCl) and subjected to rotation at 4° C. for 20minutes. Following incubation, the beads were separated from thehemoglobin-depleted sample by centrifugation. Control samples were notsubjected to this depletion step.

Protein Quantification.

The Coomassie Blue dye protein assay (BioRad Laboratories, Hercules,Calif., USA) was used for protein quantification.

Protein Enrichment and Purification.

Using the ProteomeLab™ IgY-12 LC10 column kit (Beckman Coulter, Inc.,Fullerton, Calif.), second dimension separation and immunoaffinitydepletion of 12 abundant plasma proteins (albumin, IgG, fibrinogen,transferrin, IgA, IgM, HDL, apo A-I and apo A-II, haptoglobin,α1-antitrypsin, α1-acid glycoprotein and α2-macroglobulin) was doneaccording to the manufacturer's protocol. Third dimension separation of400 μg of the enriched proteome, performed on the PF 2D platform(Beckman Coulter, Inc., Fullerton, Calif.), was done using PS-HPRP 2D(4.6×33 mm) columns (Beckman-Coulter, Inc.). Solvent A was 0.1% TFA inwater and solvent B was 0.08% TFA in acetonitrile. The gradient was runfrom 5 to 15% B in 1 min, 15% to 25% in 2 min, 25 to 31% in 2 min, 31 to41% in 10 min, 41 to 47% in 6 min, 47 to 67% in 4 min, finally up to100% B in 3 min, held for 1 min, and back to 5% in 1 min at a flow rateof 1 mL/min. The resulting 39 RP-HPLC fractions were obtained using afraction collector and 96-well plates. The fractionated proteins weredried using a speedvac system and digested with trypsin (Promega,Madison, Wis.) for MS/MS analysis.

MS Analysis for Protein Identification.

Tandem (LC-MS/MS) experiments were performed on a LTQ-Orbitrap hybridmass spectrometer (ThermoFisher, San Jose, Calif.) equipped with anon-line nano-HPLC (Agilent technologies, 1200 Series, Wilmington, Del.),as previously described. The LTQ raw data were analyzed using PASS(Integrated Analysis, Bethesda, Md.) with X!Tandem searches(www.thegpm.org; version 2008 Dec. 1) of the non-redundant InternationalProtein Index (WI) peptide database (human, 3.19). Peptideidentifications were accepted if they could be established at greaterthan 95% probability and contained at least 2 average unique identifiedspectra, with probability based Mowse scores greater than 35 (p<0.05). Achange in charge state of a peptide was not considered a uniqueidentification. The dataset was filtered to 90% sequence identity withCD-HIT.

Proteins, genes, functions and clinical associations were checked andverified using GeneCards (http://www.genecards.org/index.shtml), theNational Center for Biotechnology Information (http://www.ncbi.nlmnih.gov/entrez/query.fcgi?db=PubMed), and Online Mendelian Inheritancein Man (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM). ProteinAtlas, Genenote, GenBank, Unigene, and SwissProt databases were reviewedto identify proteins with increased expression in brain. A compositelist of brain proteins was created and used to filter the MS dataagainst to identify brain proteins in children with and without SCD.

Proteins were given a brain tissue specificity score based on theirrelative expression of transcript in human brain, liver, and 26 othernormal human tissues as assessed by available microarray, expressedsequence tags (EST), and serial analysis of gene expression (SAGE) data.Specifically, each protein was scored according to the followingcriteria: microarray data showing greater than ten-fold increase inexpression over baseline, EST and SAGE data showing presence of theprotein in less than two other tissues. Proteins received either a scoreof 1 or 0 for each category, and a maximum score of 3 was assigned ifthe protein had greater than a ten-fold increase in expression bymicroarray data, and was found in less than two tissues by EST and SAGE.

Neurogranin was chosen for validation because it showed tissuespecificity (brain tissue specificity score=3), had been previouslyimplicated in brain injury, and had the highest total spectral counts ofall the brain proteins identified.

Ingenuity Pathway Analysis.

The Ingenuity pathway analysis program (http://www.ingenuity.com) wasused to analyze the pathway network of the proteins with abundancechanges that were identified through MS. The protein accession numberswere uploaded as an Excel spreadsheet file into the Ingenuity software,which uses the data to navigate the Ingenuity pathways database andextract networks between the proteins. A score better than 2 is usuallyconsidered as a valid network.

Development of Immunoassay Protocol.

An electrochemiluminescent sandwich immunoassay was developed formeasuring Neurogranin using the MesoScale Discovery platform (MesoScaleDiscovery, Gaithersburg, Md.). The monoclonal anti-Nrgn described inExample 1 was used for the capture antibody (ab). A polyclonal anti-Nrgn(Covance, Berkeley, Calif.) and anti-species Sulfo-Tag (MesoScaleDiscovery) mixture was used for detection. The standard curve wasconstructed by serial dilution of purified Neurogranin in 1% bovineserum albumin (SeraCare Life Sciences, Milford, Mass.), from 40 ng/ml to0.055 ng/ml. Plasma spiked with Neurogranin shows an average of 99%recovery at 10 ng/mL when compared to a standard curve generated withbovine serum albumin. The lower limit of quantification, defined as thelowest dilution with a calculated concentration that does not exceed 20%of the coefficient of variation, was 0.2 ng/mL. The specificity of theassay was tested using GFAP antibody.

Final Neurogranin Assay Protocol.

Standard bind plates (MesoScale Discovery) were coated with 30 μl ofmonoclonal anti-Nrgn pool that was diluted 1:1000 withphosphate-buffered saline (PBS). The plates were incubated overnight andthen each well was blocked with 5% BSA/PBS and incubated with shaking(600 rpm) at room temperature for one hour. From a startingconcentration of 40 ng/ml, purified Neurogranin was diluted 1:3 with 1%BSA/PBS. Plasma samples were diluted 1:1 using 1% BSA/PBS, then 25 μl ofstandards and diluted sample were added in duplicate to the plate. Aftertwo hours of incubation with shaking, the plates were washed three timeswith 300 μl/well of PBS with 0.5% Tween-80 (wash buffer) using BioTek(Winooski, Vt.) automatic plate washer. The polyclonal anti-Nrgn andanti-species Sulfo-Tag were diluted 1:1000 with 1% BSA/PBS to give afinal concentration of 1 μg/mL. Subsequently, 25 μl were added to eachwell, and each plate was incubated with shaking for one hour beforebeing washed three times. Plates were then read with a Sector Imager2400 (MesoScale Discovery).

Recombinant Neurogranin Protein Production.

A PCR cloning strategy was used to clone Neurogranin cDNA into bacteriaexpression vectors to express recombinant proteins. The primers weredesigned based on human Neurogranin cDNA sequence, SgfI (in forwardprimer) and MluI (in reverse primer) cutting sites were introduced in tothe primers respectively to ensure the correct reading frame and cloningconvenience. The sequences for primers are: forward5′-GAGGCGATCGCCATGGACTGCTGCACCGAGAAC-3′ (SEQ ID NO:9) and Reverse5′-GCGACGCGTCTAGTCTCCGCTGGGGCCGC-3′ (SEQ ID NO:10). Human NeurogranincDNA (Origene, RC201209) was used as template for PCR amplification. ThePCR product was digested with SgfI and MluI, gel purified and ligatedinto pre-digested vectors pEX-N-His-GST (Origene, PS100028) andpEX-N-His (Origene, PS100029) respectively. DH5α competent cells(Invitrogen, 18265017) were used for transformation and plasmid DNApropagation. Positive clones were identified by restriction enzymesdigestion analysis and further confirmed by DNA sequencing. The properplasmids containing correct Neurogranin cDNA were then used to transformRosetta (Novagen) strain for protein expression. Single positive Rosettaclone was grown in Overnight Express Instant TB media (Novagen, #71491)at 37° C. overnight. Ni-NTA Superflow Columns (Qiagen, #30622) was usedto purify His-Nrgn protein, Glutathione Spin Column (Pierce, #16104) wasused to purify His-GST-Nrgn protein; all the purification procedureswere followed manufacturer's instructions. The samples of recombinantproteins were subjected to SDS-PAGE and visualized by coomassiestaining; the protein bands were then isolated, digested and confirmedby mass spectrometry analysis.

Monoclonal Antibody Production.

Mouse anti-Nrgn monoclonal antibody was produced at The MonoclonalAntibody Core Facility (MACF) at Johns Hopkins University, Department ofNeuroscience. Briefly, five 6 weeks old BALB/c female mice (CharlesRiver, Wilmington, Mass.) were immunized with intraperitoneal injectionof 100 ug of His-Nrgn and boosted on day 21 with using a same route.Then the mice were subcutaneous injection with 50 ug of His-Nrgn inIncomplete Freund's Adjuvant twice. Blood was collected 10 daysfollowing the third immunization. The sera were tested by direct ELISAusing His-GST-Nrgn protein as target protein. The mouse with best titerin the ELISA was selected for a final i.v boost. On day 89, the mousewas sacrificed and the spleen was removed for the fusion process.

Polyethylene glucol (MW1500, Sigma) was used to fuse P3×653.Ag8 mousemyeloma cells with spleen cells from the immunized mouse. Fused cellswere distributed in 96-well tissue culture plate containing feeder cellsharvested from the peritoneum of a normal BALA/c mouse primedintraperitoneally with 0.5 ml Incomplete Freund Adjuvants 4 daysearlier. After 10 days of undisturbed culture in selection medium (DMEMcontaining 20% FCS, Hyclone, supplemented 1×OPI (Sigma), 100 uMhypoxanthine, 0.4 uM aminopterin, and 160 uM thymidine), supernatantswere tested with ELISA as described above. Positive colonies were clonedtwice by limiting dilution on splenocytes from normal BALA/c mice asfeeder cells.

The cloned hybridoma line (30.5.2) was grown in DMEM containing 10%defined FCS (Hyclone) supplemented with 1×OPI for 4 to 6 days. Then,hybridoma cells were adapted to growth in serum free media, which wouldhave allowed antibody production in the in vitro system. When cells werein log growth phase, 2×10⁷ cells were inoculated into the in vitrosystem. The culture supernatant (antibody) was collected every fivedays.

Statistics.

The differences in clinical characteristics between groups were assessedusing two-tailed t-tests with unequal variance. The differences betweengroups for Neurogranin were compared using the non-parametricMann-Whitney U test. The Kruskal-Wallis test was used to compare medianplasma levels across the three groups: SCD with SCI, SCD without SCI,and healthy controls. Correlations between Neurogranin concentrationsand other variables were estimated according to Pearson. In theanalyses, a P value less than 0.05 was considered statisticallysignificant. Statistical analyses were conducted using Stata version11.0 (StataCorp. College Station, Tex.).

Results

Baseline Characteristics of Children with SCD and Controls.

As expected, comparisons of the plasma samples used for proteomicanalysis showed that children with SCD (n=15) have significantly lowerbaseline hb and hematocrit (hct) values as compared to their healthynon-SCD counterparts (n=6) (Table 1).

TABLE 1 Clinical Characteristics of Children with SCD and Healthy,Non-SCD Controls SCD Non-SCD (n = 15) (n = 6) % SCT (n) — 50 (3)  % Male(n) 67 (10) 50 (3)  Age 9.4 (2.7) 11.5 (2.1)  Retic (%)^(A) 9.5 (2.7)0.9 (0.3) Hb (g/dL)^(A) 8.5 (0.8) 12.3 (0.9)  Hct (%)^(A) 23.9 (2)    37(2.5) WBC (×10⁹/L)^(A) 14.9 (5.5)  5.5 (1.4) Plt (×10⁹/L)^(A) 463 (126)330 (19)  Results represent mean ± standard deviation. SCT, sickle celltrait, Retic, reticulocyte; Hb, hemoglobin; Hct, hematocrit; WBC, whiteblood cell count; Plt, platelet. ^(A)P < 0.05 between groups byStudent's t-test.

WBC and reticulocyte counts were significantly higher in children withSCD. Table 2 shows the baseline demographic information for SCD childrenwith and without SCI that were used for proteomic analysis (n=7 SCI, 8non-SCI) and for the quantification of Neurogranin (n=65 SCI, 51non-SCI). There were no significant differences in baseline hb, WBC,platelet or reticulocyte counts. Patients with SCI had higher levels oftotal bilirubin as compared to SCD children without SCI.

TABLE 2 Clinical Characteristics of Children with SCD For ProteomicAnalysis For Protein Quantification (n = 15) (n = 115) SCI Non-SCI SCINon-SCI (n = 7) (n = 8) (n = 65) (n = 51) % Male 71 (5) 63 (5) 53 (34)41 (21) (n) Age 9.8 (2.4) 9 (3.1) 9.8 (3.1) 8.9 (2.9) Retic (%) 11 (2.108.2 (2.6) 11 (4.8) 10.6 (3.8) Hb (g/dL) 8.3 (0.89) 8.6 (0.7) 8.3 (1.1)8.6 (1.2) Hct (%) 23.3 (2.3) 24 (1.8) 23.6 3.5) 24.5 (3.6) WBC 15.8(7.5) 14.1 (3.1) 12.1 (4.6) 13.4 (10.1) (×10⁹/L) Plt 410 (79) 510 (145)453 (143) 435 (164) (×10⁹/L) TBili^(A) 5.6 (3.4) 2.7 (0.9) 3.7 (2.4) 2.9(1.6) Results represent mean (±standard deviation) (SD). Retic,reticulocyte; Hb, hemoglobin; Hct, hematocrit; WBC, white blood cellcount; Plt, platelet; TotBili, total bilirubin. ^(A)P < 0.05 betweengroups by Student's t test.

Characterization of the Plasma Proteome of Children with SCD.

Using three sequential separation steps of Hb removal, immunoaffinityfractionation, and HPLC separation, followed by tandem MS and X! Tandemsearches of the IPI peptide database, 752 proteins were identified inthe SCI group and an additional 390 proteins were identified in thenon-SCI group, totaling 1162 unique proteins circulating in the plasmaproteome of children with SCD. An additional 239 proteins wereidentified in healthy controls (n=639 total proteins in non-SCDchildren). Thirty percent (343/1162) of the proteins identified inchildren with SCD were seen in the SCI group and not in the non-SCDgroup.

Analysis using ingenuity pathway software revealed that the proteinsidentified in both SCD and normal controls demonstrated significantoverrepresentation of a number of biological functions and pathways,including cell-to-cell signaling and cell death, immune celltrafficking, and acute phase response signaling. Neurological diseasewas ranked amongst the top five diseases in the SCI and normal groups,but not in the non-SCI group. Further analysis revealed that theproteins involved in the SCI group pathways are involved in morespecific disease processes that have already been implicated in sicklecell disease, namely ischemia-reperfusion injury, endothelialdysfunction and neuronal injury and death. Furthermore, in children withSCD and SCI the specific conditions related to neurologic diseasediffered from the normal controls in that proteins associated withtauopathy (microtubule-associated protein tau, glial fibrillary acidicprotein) and cerebral amyloid angiopathy (cystatin C, vimentin) wereseen in the SCI group only. In addition, while loss of neurites wereseen in both the SCI and normal groups, only the SCI group had proteinsassociated with loss of axons (microtubule-associated protein tau),suggesting neuronal injury in addition to cell death.

Identification of Brain Proteins.

The MS data was filtered against a list of proteins with increasedprotein expression in brain to produce a composite list of brainproteins that are found in children with SCD. Using this methodology, atotal of twenty-seven brain proteins were identified in children withSCD (data not shown). None of these proteins were identified in non-SCDcontrols. Among the brain proteins identified, Neurogranin had greaterthan a ten-fold increase in expression in the brain and was found inless than two tissues by EST and SAGE. It also had the highestconfidence score and total number of spectral counts.

Plasma Neurogranin Levels in Children.

Neurogranin, a calcium-sensitive calmodulin-binding neuron-specificprotein that has been implicated in synaptic development and remodeling,was found in SCD children with and without SCI. As shown in FIG. 9A,median plasma Neurogranin levels were significantly higher in childrenwith SCD (0.72 μg/ml) as compared to non-SCD controls (0.12 μg/ml,P<0.001). Among children with SCD, sixty-three percent of SCI (40/64)and non-SCI (32/51) children had median Neurogranin values that wereabove the 95^(th) percentile for non-SCD values (FIG. 9B). There were nodifferences in median Neurogranin levels between the SCI (0.69 μg) andnon-SCI groups (0.73, P=0.6, data not shown). Neurogranin did notcorrelate with known risk factors for SCI, including hb, hct, WBC,platelet, and reticulocyte counts, systolic blood pressure and percentof hb F.

Discussion

Biomarkers of subclinical brain injury in SCD are needed to diagnosisand aid in the development of molecular targeted therapies, as well asto monitor disease response. Proteomics provides an opportunity todiscover these biochemical markers in complex mixtures, such as plasma.However, an established methodology for the discovery of brainbiomarkers in children with SCD has not been previously reported. Aproteomic-based approach was used to test the hypothesis that childrenwith SCD and SCI have brain proteins circulating in their plasmaproteome that is associated with subclinical brain injury. Using aworkflow of sequential depletion steps, followed by fractionation byRP-HPLC and label-free quantification on a LTQ-Orbitrap massspectrometer, 1162 proteins were identified and characterized in thechildren with SCD, of which twenty-seven were found to have highexpression in the brain. The experimental approach was validated usingan immunoassay developed to measure Neurogranin.

Neurogranin was measured in children with SCD and age-matched, non-SCDcontrols and found that children with SCD had significantly higherplasma levels of Neurogranin. In fact, greater than sixty percent of SCDchildren, with and without SCI, had Neurogranin values that were greaterthan the 95^(th) percentile for observed Neurogranin values in non-SCDcontrols. Neurogranin has not been previously studied in children withSCD. In fact, previous studies have largely been done in adults withschizophrenia and Alzheimer disease, and have related Neurogranin toimpairments in learning and memory. Neurocognitive deficits in academicachievement and memory in children with SCD have been well documented.When compared with siblings and age-matched peers, school-aged SCDchildren with stroke appeared to have more neuropsychological deficits,but deficits were also found in patients who had clinically milddisease. These findings of increased Neurogranin in children with SCDmay be relevant to the etiology of neurocognitive dysfunction, asmeasured by IQ, that is observed in these patients. Further studies toevaluate whether existing therapies, such as blood transfusions andhydroxyurea, modulate levels of Neurogranin would be informative, andmay provide a measurable way to evaluate whether these therapies canhelp maintain brain function, or perhaps even reverse any loss offunction.

In addition, the results from pathway analysis confirm that childrenwith SCD are at risk for neuronal injury and cell death, and suggestspecific mechanisms for injury including tauopathy and axonal loss. Theanalysis suggested that glial fibrillary acidic protein (GFAP), anintracellular intermediate filament protein that is a known biomarker ofstroke, is associated with tauopathy in children with SCD and SCI. Thepresent inventors have previously shown that GFAP is elevated inpatients with SCD when compared to healthy controls, and is associatedwith ischemic brain injury, including silent cerebral infarctions.

In summary, the present inventors have developed and verified aproteomic work-flow for brain biomarker discovery in children with SCD.The present inventors are the first to report significant elevations ofNeurogranin in children with SCD as compared to age-matched, non-SCDcontrols, and provide additional insight into the pathophysiology ofsubclinical brain injury. Collectively, these findings support clinicalinvestigation for biomarker discovery in children with SCD and SCI, andprovide new potential targets for therapeutic drug discovery.

1. An isolated antibody or fragment thereof that specifically binds toneurogranin.
 2. An isolated antibody or fragment thereof thatspecifically binds to amino acids 1-78 of SEQ ID NO:11.
 3. An isolatedantibody or fragment thereof that specifically binds to amino acids55-78 of SEQ ID NO:11.
 4. The isolated antibody or fragment thereof ofclaim 1, wherein the antibody or fragment thereof is polyclonal.
 5. Theisolated antibody or fragment thereof of claim 1, wherein the antibodyor fragment thereof is monoclonal.
 6. The isolated antibody or fragmentthereof of claim 1, wherein the antibody or fragment thereof ismammalian.
 7. The isolated antibody or fragment thereof of claim 1,wherein the antibody or fragment thereof is human.
 8. A hybridoma cellwhich produces the antibody or fragment thereof according to claim
 5. 9.The isolated antibody or fragment thereof of claim 1, wherein thefragment is selected from the group consisting of a Fab fragment; aF(ab′) 2 fragment; a Fv fragment; and a single chain fragment.
 10. Theisolated antibody or fragment thereof of claim 1, further comprising adetectable substance coupled to the antibody.
 11. The isolated antibodyor fragment thereof of claim 10, wherein the detectable substance isselected from the group consisting of an enzyme; a fluorescent label; aradioisotope; and chemiluminescent label.
 12. The isolated antibody orfragment thereof of claim 10, which specifically binds to neurogranin inan ELISA.
 13. The isolated antibody or fragment thereof of claim 10,which specifically binds to neurogranin in a competitive-binding assay.14. The isolated antibody or fragment thereof of claim 10, whichspecifically binds to neurogranin in a radioimmunoassay.
 15. Theisolated antibody or fragment thereof of claim 10, which specificallybinds to neurogranin in a fluorescence-activated cell sorting (FACS)assay.
 16. A kit for detecting neurogranin comprising: (a) the isolatedantibody of claim 1; and (b) at least one component to detect binding ofthe isolated antibody to neurogranin.
 17. An isolated antibody obtainedfrom an animal that has been immunized with neurogranin, wherein theantibody specifically binds to an antigenic epitope-bearing polypeptidefragment of neurogranin.
 18. A monoclonal antibody designated 30.5.2that specifically binds neurogranin.
 19. An anti-neurogranin monoclonalantibody which is produced by the hybridoma designated 30.5.2.
 20. Apolynucleotide aptamer that specifically binds neurogranin.
 21. Thepolynucleotide aptamer of claim 20, wherein the neurogranin is humanneurogranin.
 22. The polynucleotide aptamer of claim 20, wherein theaptamer binds to neurogranin with a Kd of less than about 1000 nM. 23.The polynucleotide aptamer of claim 20, wherein the aptamer binds toneurogranin with a Kd of less than about 100 nM.
 24. The polynucleotideaptamer of claim 20, wherein the aptamer binds to neurogranin with a Kdof less than about 20 nM.
 25. The polynucleotide aptamer of claim 20,which consists of about 10 to about 100 nucleotides.
 26. Thepolynucleotide aptamer of claim 25, which consists of about 20 to about80 nucleotides.
 27. The polynucleotide aptamer of claim 26, whichconsists of about 30 to about 50 nucleotides.
 28. The polynucleotideaptamer claim 20, which is an RNA aptamer.
 29. The polynucleotideaptamer of claim 28, comprising a nucleotide sequence at least 80%identical to any one of SEQ ID NOS: 4-6 or a fragment thereof of atleast ten contiguous nucleotides.
 30. The polynucleotide aptamer ofclaim 29, comprising a nucleotide sequence at least 90% identical to anyone of SEQ ID NOS: 4-6 or a fragment thereof of at least ten contiguousnucleotides.
 31. The polynucleotide aptamer of claim 30, comprising anucleotide sequence at least 95% identical to any one of SEQ ID NOS: 4-6or a fragment thereof of at least ten contiguous nucleotides.
 32. Thepolynucleotide aptamer of claim 31, comprising the nucleotide sequenceof any one of SEQ ID NOS: 4-6 or a fragment thereof of at least tencontiguous nucleotides.
 33. The polynucleotide aptamer of claim 31,consisting of the nucleotide sequence of any one of SEQ ID NOS: 4-6 or afragment thereof of at least ten contiguous nucleotides.
 34. Thepolynucleotide aptamer of claim 20, further comprising at least onemodified internucleotide linker.
 35. The polynucleotide aptamer of claim20, comprising at least one terminal blocker.
 36. The polynucleotideaptamer of claim 20, which is linked to a conjugate.
 37. Apolynucleotide encoding the polynucleotide aptamer of claim
 20. 38. Avector comprising the polynucleotide of claim
 37. 39. A cell comprisingthe polynucleotide aptamer of claim
 20. 40. (canceled)
 41. A cellcomprising the polynucleotide of claim
 37. 42. A cell comprising thevector of claim
 38. 43. A method of diagnosing a disease or disorderassociated with neurogranin in a subject, comprising measuring the levelof neurogranin in the subject by binding neurogranin with thepolynucleotide aptamer of claim 20 and determining the amount of aptamerbound to neurogranin.
 44. The method of claim 43, wherein the bindingoccurs in a sample obtained from the subject.
 45. A method fordetermining the amount of neurogranin in a sample comprising the step ofdetecting a peptide specific to neurogranin using a triple quadrupolemass spectrometer and multiple reaction monitoring, wherein the peptidespecific to neurogranin comprises SEQ ID NO:7.